Structural analyses of the group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations.

Manenda MS, Picard MÈ, Zhang L, Cyr N, Zhu X, Barma J, Pascal JM, Couture M, Zhang C, Shi R, J Biol Chem (2020) Europe PMC

SASDHC2 – PieE-FAD: 2,4-dichlorophenol 6-monooxygenase bound to flavin adenine dinucleotide

2,4-dichlorophenol 6-monooxygenase
Flavin adenine dinucleotide
MWexperimental 390 kDa
MWexpected 404 kDa
VPorod 624 nm3
log I(s) 1.04×10-1 1.04×10-2 1.04×10-3 1.04×10-4
2,4-dichlorophenol 6-monooxygenase Flavin adenine dinucleotide small angle scattering data  s, nm-1
ln I(s)
2,4-dichlorophenol 6-monooxygenase Flavin adenine dinucleotide Guinier plot ln 1.04×10-1 Rg: 4.8 nm 0 (4.8 nm)-2 s2
(sRg)2I(s)/I(0)
2,4-dichlorophenol 6-monooxygenase Flavin adenine dinucleotide Kratky plot 1.104 0 √3 sRg
p(r)
2,4-dichlorophenol 6-monooxygenase Flavin adenine dinucleotide pair distance distribution function Rg: 4.7 nm 0 Dmax: 13.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
2,4-dichlorophenol 6-monooxygenase Flavin adenine dinucleotide DAMMIN model
SAXS data from solutions of PieE-FAD in 20 mM Tris, 150 mM NaCl, 5 mM DTT, 2% glycerol, pH 7.5 were collected using a Xenocs BioXolver L with MetalJet (Université de Montréal) equipped with a Pilatus3 R 300K detector at a sample-detector distance of 0.6 m and at a wavelength of λ = 0.134 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: The PieE-FAD sample (400 µl at 17 mg/ml) was injected onto a GE Healthcare Superdex 200 Increase 10/300 column. Elution took place at a flow rate of 0.1 ml/min at 23 °C. A total of 190 SAXS data frames, each exposed for 30 seconds, were recorded at 20 °C during the size-exclusion chromatography. After background buffer (frames 40 to 60) and baseline (frames 10-20 to 179-189) corrections, SEC-SAXS data frames 73 to 84, corresponding to the elution peak at half-height of the peak, were scaled, averaged and binned logarithmically.

2,4-dichlorophenol 6-monooxygenase (PieE)
Mol. type   Protein
Organism   Streptomyces sp. SCSIO 03032
Olig. state   Hexamer
Mon. MW   66.6 kDa
 
UniProt   W0C4C9 (1-598)
Sequence   FASTA
 
Flavin adenine dinucleotide (FAD)
Mol. type   Other
Olig. state   Hexamer
Mon. MW   0.8 kDa
Chemical formula